The present invention relates generally to optical instrumentation and relates more particularly to the beam-steering of light using acousto-optical deflectors.
Optical instruments have long played an important role in the study of physical and biological phenomena. Light microscopes, in particular, have been used for more than one hundred years to gain insight into the structure of biological media. As can readily be appreciated, achieving high spatial resolution remains one of the foremost objectives of a light microscope. This objective, however, is often hampered by the fact that biological media, by their very nature, are typically highly scattering with respect to light. Consequently, objects located beneath the surface of a biological medium are often difficult to observe with high resolution using light microscopy. As a result, a number of different approaches have been undertaken in an effort to counteract the light scattering effects of biological media.
One such approach is the confocal microscope, an example of which is disclosed in U.S. Pat. No. 4,863,226, inventors Houpt et al., which issued Sep. 5, 1989, and which is incorporated herein by reference. In a confocal microscope, light is brought to focus on or within a sample, and the light emitted from the illuminated sample is then brought to focus on a pinhole positioned in front of a detector, the pinhole being used to prevent light scattered by the sample from reaching the detector. Often in a confocal microscope, the illuminating light is laser light, and a galvanometer or the like is placed along the optical path of the illuminating laser light to create a scanning beam of illuminating laser light. Laser scanning confocal microscopes are often used to create fluorescence images of a sample, with the illuminating laser light being used to excite native and/or extrinsic fluorophores present within the sample, and the non-scattered component of the fluorescent light emitted from said fluorophores being passed through the pinhole and detected by the detector.
One of the problems associated with the use of laser scanning confocal microscopes in fluorescence imaging is that the detected light signal is typically weak. This is because, of all the fluorescence photons generated by the sample, only the non-scattered (i.e., ballistic) photons generated at the illuminating focus (i.e., on-focus) are permitted to pass through the pinhole and are detected by the detector. In other words, not only are the undesirable fluorescence photons generated at loci other than the illuminating focus (i.e., off-focus) excluded from detection but so are the desirable scattered on-focus fluorescence photons, said scattered on-focus fluorescence photons representing a significant portion of the on-focus fluorescence photons.
Another problem associated with the use of laser scanning confocal microscopes in fluorescence imaging is that the intensity of the illuminating light necessary to generate an appreciable detected signal often has the undesirable consequence of adversely affecting the fluorophore (i.e., photobleaching) or adversely affecting the sample through a fluorophore-mediated event (i.e., photodamage). Moreover, because the illuminating light must travel through the sample to the illuminating focus, the above-mentioned effects of photobleaching and photodamage are not confined to the illuminating focus.
One form of laser scanning microscopy that has been devised to address the types of shortcomings discussed above in connection with laser scanning confocal fluorescence microscopy is multi-photon excited fluorescence laser scanning microscopy. In multi-photon excited fluorescence laser scanning microscopy, excitation of a fluorophore is achieved by the simultaneous absorption by the fluorophore of two or more photons of low energy that combine their energies to provide the requisite energy for transition of the fluorophore to its excited state. For example, two photons of lower energy red or infrared light may be used to excite a fluorophore typically excitable by one photon of higher energy ultraviolet light. Because multi-photon absorption requires two or more photons for each excitation, its rate depends on the square of the instantaneous intensity and is, therefore, almost completely confined spatially to the high-intensity region at the focal point of the strongly focused excitation laser.
Consequently, because the requisite energy for excitation is spatially confined to the focal point of the illuminating laser, multi-photon excited fluorescence laser scanning microscopy does not result in off-focus photobleaching or photodamage and does not require the placement of a pinhole in front of the detector, as in confocal fluorescence microscopy, to reject off-focus fluorescence photons. Because such a pinhole is unnecessary in multi-photon excited fluorescence laser scanning microscopy, both scattered and ballistic on-focus fluorescence photons are detected, thereby yielding a stronger signal than if only ballistic on-focus fluorescence photons were detected. Furthermore, because longer wavelength photons typically scatter less in biological media than do shorter wavelength photons, one can achieve improved depth penetration of the media using multi-photon excited fluorescence laser scanning microscopy than using laser scanning confocal fluorescence microscopy.
Additional information relating to multi-photon excited fluorescence laser scanning microscopy is provided in the following published documents, all of which are incorporated herein by reference: U.S. Pat. No. 5,034,613, inventors Denk et al., which issued Jul. 23, 1991; Denk et al., xe2x80x9cTwo-Photon Laser Scanning Fluorescence Microscopy,xe2x80x9d Science, 248:73-6 (1990); Denk et al., xe2x80x9cPhoton Upmanship: Why Multiphoton Imaging Is More than a Gimmick,xe2x80x9d Neuron, 18:351-7 (1997); Denk et al., xe2x80x9cTwo-Photon Molecular Excitation in Laser-Scanning Microscopy,xe2x80x9d Handbook of Biological Confocal Microscopy, pages 445-57, edited by James B. Pawley, Plenum Press, New York (1995); Fan et al., xe2x80x9cVideo-Rate Scanning Two-Photon Excitation Fluorescence Microscopy and Ratio Imaging with Cameleons,xe2x80x9d Biophysical Journal, 76:2412-20 (1999); Koester et al., xe2x80x9cCa2+ Fluorescence Imaging with Pico- and Femtosecond Two-Photon Excitation: Signal and Photodamage,xe2x80x9d Biophysical Journal, 77:2226-36 (1999); Mainen et al., xe2x80x9cTwo-Photon Imaging in Living Brain Slices,xe2x80x9d METHODS: A Companion to Methods in Enzymology, 18:231-9 (1999); and Parthenopoulos et al., xe2x80x9cThree-Dimensional Optical Storage Memory,xe2x80x9d Science, 245:843-5 (1989).
Another form of laser scanning microscopy that has been devised to address the types of shortcomings discussed above in connection with laser scanning confocal fluorescence microscopy is multi-harmonic generation laser scanning microscopy. In one type of multi-harmonic generation laser scanning microscopy, namely, second-harmonic generation laser scanning microscopy, the combined coherent electric fields of two incident photons interact with a dipolar molecule. The incident field is scattered and, in the process, a single photon of exactly half the incident photon wavelength and twice the incident photon energy is formed instantly. This photon is then detected.
As a second-order reaction in the concentration of incident photons, second-harmonic generation laser scanning microscopy possesses the same intrinsic resolving power as two-photon excited fluorescence laser scanning microscopy. In addition, second-harmonic generation laser scanning microscopy, like multi-photon excited fluorescence laser scanning microscopy and unlike laser scanning confocal fluorescence microscopy, does not require the placement of apinhole in front of the detector. However, unlike multi-photon excited fluorescence laser scanning microscopy, multi-harmonic generation laser scanning microscopy does not require that the object being imaged possess a fluorescent molecule. Instead, multi-harmonic generation laser scanning microscopy merely requires that the object possess the appropriate nonlinear susceptibility. Moreover, as contrasted with multi-photon excited fluorescence laser scanning microscopy, multi-harmonic generation laser scanning microscopy does not involve the absorption and re-emission of energy as only scattering occurs therein and it occurs instantly. Yet another difference between multi-photon excited fluorescence laser scanning microscopy and multi-harmonic generation laser scanning microscopy is that the latter technique requires the use of forward-scattered detection and collection optics since harmonic light propagates only in the forward direction with respect to the exciting light whereas the former technique does not require such forward placement as it relies upon fluorescent light, which is radiated isotropically.
Additional information relating to multi-harmonic generation laser scanning microscopy is provided in the following published documents, both of which are incorporated herein by reference: Campagnola et al., xe2x80x9cHigh-Resolution Nonlinear Optical Imaging of Live Cells by Second Harmonic Generation,xe2x80x9d Biophysical Journal, 77:3341-9 (December 1999); and Moreaux et al., xe2x80x9cCoherent Scattering in Multi-Harmonic Light Microscopy,xe2x80x9d Biophysical Journal, 80(3):1568-74 (March 2001).
Acousto-optical deflectors are devices commonly used in the high-speed scanning of light beams. An acousto-optical deflector typically comprises a solid transparent block of homogenous material (e.g., TeO2) onto which one or more rf transducers are bonded. The transducers produce acoustic plane waves that travel through the block of homogeneous material and, thereby, cause a periodic refractive index modulation within the block. Due to the large difference in the respective velocities of sound and light, incident light xe2x80x9cseesxe2x80x9d this refractive index modulation as a stationary grating and is deflected at a specific angle (the so-called xe2x80x9cBragg anglexe2x80x9d) with respect to the acoustic wave. As seen by the following equation, the deflection of the incident beam is proportional to the frequency of the acoustic wave (and, thus, the period of the refractive index modulation):                     θ        =                              2            ·                          θ              Bragg                                =                                    2              ·                                                λ                  ⁢                                      xe2x80x83                                    ⁢                  f                                                  2                  ⁢                                      xe2x80x83                                    ⁢                  v                                                      =                                          λ                ⁢                                  xe2x80x83                                ⁢                f                            v                                                          [                  Eq          .                      xe2x80x83                    ⁢          1                ]            
where xcex8=deflection angle with respect to the incident beam [radians], xcex=wavelength of light, f=acoustic wave frequency, and v=velocity of the acoustic wave.
A linear scan of an incident beam by an acousto-optical deflector of the type described above can be produced by ramping the frequency of the rf signal that drives the transducers. Other patterns of beam deflection by an acousto-optical deflector, such as movement of the beam through a set of predefined positions, without an intervening sweep (the so-called xe2x80x9crandom accessxe2x80x9d steering), are also possible by applying appropriate command functions to the transducers.
Because acousto-optical deflectors function without moving parts, imaging systems that use acousto-optical deflectors to generate scanning beams possess certain advantages over imaging systems that use movable deflection mirrors to generate scanning beams. More specifically, imaging systems that use acousto-optical deflectors can acquire images at rates from about 30 to nearly 500 Hz and are anticipated to operate at even higher (kHz) repetition rates in random access mode. By contrast, if control of the deflection mirrors involves feedback with a linear command function, the line scan frequency of the deflection mirrors is typically less than 500 Hz. This results in a maximal image acquisition rate of about 1 Hz, a serious limitation to real-time analysis. Higher scan frequencies can be attained when the mirrors are made to be freely oscillating without feedback, but then the mirror deflections become essentially sinusoidal, instead of linear. This has the drawback that only a fraction of the working cycle of a full deflection period (the fraction in which the sine function is approximately linear) is available for data acquisition or that significant post-acquisition processing is required to linearize the image. In addition, the inert mass of a scanning deflection mirror precludes the abrupt accelerations and decelerations that would be required for random access.
Because acousto-optical deflectors of the type described above permit laser scanning at high repetition rates, it would seem to be desirable to utilize such acousto-optical deflectors for laser scanning in multi-photon laser scanning microscopy. However, this has not been feasible because the ultrashort laser pulses that are needed for multi-photon excitation (and for multi-harmonic generation) at biologically tolerable average power levels are not typically monochromatic, but rather, span spectral ranges of up to tens of nanometers. As a result, because the incident ultrashort light pulses on an acousto-optical deflector are typically multi-chromatic, the acousto-optical deflector acts essentially as a diffraction grating for the incident ultrashort light pulses, laterally separating their spectral components (see FIG. 1A). Consequently, pulses with a center wavelength of 900 nm and a bandwidth of about 20 nm, as is typical for the 100 fs pulses used in multi-photon laser scanning microscopy (and in multi-harmonic generation laser scanning microscopy), are dispersed by 0.0729 degrees using an acousto-optical deflector having an acoustic velocity of 4322 m/s and a frequency range centered at 275 MHz. This corresponds to a normalized dispersion of 0.00365 deg/nm. Since a linear scan spans a range of acoustic frequencies, the normalized dispersion usually varies slightly across the scan area, as shown in TABLE I.
In view of the above, it can readily be appreciated that the lateral dispersion of ultrashort laser pulses by an acousto-optical deflector blurs the focus of the exciting beam in the scan direction, with the following two adverse consequences for multi-photon laser scanning microscopy: (i) the spatial resolution in the scan direction is severely compromised; and (ii) the multi-photon excitation efficiency (and the multi-harmonic generation efficiency) is lowered.
It is an object of the present invention to provide a technique for ameliorating the above-described problem of spectral dispersion of multi-chromatic ultrashort light pulses by acousto-optical deflectors.
Such a technique is provided, in accordance with the teachings of the present invention, by positioning a spectrally dispersive element, such as a prism, along the path of the multi-chromatic ultrashort light pulses in such a way that said dispersive element disperses the multi-chromatic ultrashort light pulses in a direction opposite to the spectral dispersion caused by the acousto-optical deflector. Preferably, the opposing dispersion provided by the spectrally dispersive element equals that provided by the acousto-optical deflector for at least a portion of the dispersed light.
According to one aspect of the invention, there is provided an apparatus for steering a beam of light, said apparatus comprising (a) an acousto-optical deflector; and (b) a spectrally dispersive element, said spectrally dispersive element and said acousto-optical deflector being optically coupled to one another. The spectrally dispersive element may be positioned either in front of said acousto-optical deflector or behind said acousto-optical deflector, said spectrally dispersive element preferably being oriented relative to said acousto-optical deflector so that said spectrally dispersive element disperses multi-chromatic light in a direction opposite to that dispersed by said acousto-optical deflector, said spectrally dispersive element also preferably being constructed to disperse multi-chromatic light in an amount equally opposite to, for at least a portion of said multi-chromatic light, that dispersed by said acousto-optical deflector.
The spectrally dispersive element is preferably a prism but may alternatively be a grating or a second acousto-optical deflector. The apparatus may further comprise one or more mirrors for use in directing the beam along a particular path. More specifically, where the spectrally dispersive element is positioned in front of said acousto-optical deflector, said apparatus preferably further comprises a rotatable mirror and a fixed mirror, said rotatable mirror and said fixed mirror being positioned between spectrally dispersive element and said acousto-optical deflector and serving to redirect the beam, after it passes through the spectrally dispersive element, to the entrance axis of the acousto-optical deflector. The rotatable mirror imparts adjustability for wavelength-dependent variations in the deflection of light by the spectrally dispersive element.
A second set of acousto-optical deflector and spectrally dispersive element can be used, for example, to steer the beam along a second axis perpendicular to the first axis.
The above-described beam steering apparatus can be used to scan a plurality of contiguous locations or can be used to randomly deflect the beam within a plurality of possible locations.
According to another aspect of the invention, there is provided a method of steering a beam of light, said method comprising the steps of (a) providing a beam of light; (b) passing said beam of light through a spectrally dispersive element; and (c) deflecting said beam of light using an acousto-optical deflector. Preferably, the spectrally dispersive element is oriented to disperse multi-chromatic light in a direction opposite to that dispersed by said acousto-optical deflector, and said spectrally dispersive element is preferably constructed to disperse multi-chromatic light, for at least a portion of said multi-chromatic light, in an amount equal to that dispersed by said acousto-optical deflector. The light may be passed through the spectrally dispersive element either before or after being deflected by the acousto-optical deflector.
The beam of light steered by the present method may be either a continuous beam of light or a pulsed beam of light. Preferably, the beam of light is a beam of ultrashort laser light pulses having a pulse duration of less than one picosecond and a bandwidth of no more than about 40 nm. Said light preferably has a wavelength in the range of about 400 to 1000 nm.
The present invention is also directed to an apparatus for spectrally dispersing multi-chromatic light traveling along a first axis, said apparatus comprising (a) a spectrally dispersive element, disposed along said first axis, for dispersing said multi-chromatic light; (b) a pair of mirrors optically coupled to said spectrally dispersive element and positioned thereafter to redirect said dispersed light along said first axis. Preferably, one of said pair of mirrors is a rotatable mirror to adjust for wavelength-dependent variations in the deflection of said dispersed light, and the other of said pair of mirrors is a fixed mirror.
The aforementioned apparatus preferably further comprises means for rotating said rotatable mirror, said rotating means comprising a rotatably mounted arm and a motor for rotating said rotatably mounted arm, said rotatable mirror being fixedly mounted on said rotatably mounted arm. Preferably, said motor is controllable by computer. Said apparatus preferably further comprises a base, said spectrally dispersive element, said pair of mirrors, said rotatably mounted arm and said motor being mounted on said base.
The present invention is additionally directed to a method of imaging a sample using multi-photon excited fluorescence laser scanning microscopy, said method comprising the steps of (a) providing a sample containing fluorescent molecules which radiate photons of a first characteristic energy; (b) producing a scanning beam of ultrashort laser light pulses, said scanning beam being produced by (i) providing a beam of ultrashort laser light pulses comprising photons of a second characteristic energy, wherein said second characteristic energy is less than said first characteristic energy and wherein the simultaneous absorption of a plurality of said photons of said second characteristic energy by said fluorescent molecules causes the fluorescence of said fluorescent molecules, (ii) passing said beam through a spectrally dispersive element, and (iii) deflecting said beam using an acousto-optical deflector, (iv) wherein said spectrally dispersive element is oriented to disperse multi-chromatic light in a direction opposite to that dispersed by said acousto-optical deflector; (c) focusing said scanning beam at a focal point within said sample to produce an illumination intensity sufficiently high only at said focal point to produce molecular excitation and fluorescence of said sample by simultaneous absorption of a plurality of incident photons; (d) detecting the fluorescence produced by said sample; and (e) using the detected fluorescence to form an image of the sample.
Preferably, the spectrally dispersive element used in the aforementioned method is constructed to disperse multi-chromatic light, for at least a portion of said multi-chromatic light, in an amount equal to that dispersed by said acousto-optical deflector. The scanning beam producing step described above preferably further comprises scanning the sample in a direction perpendicular to said first axis, said scanning in a direction perpendicular to said first axis comprising the use of a scanning mirror or a second acousto-optical deflector and a second spectrally dispersive element, said second spectrally dispersive element being oriented relative to said second acousto-optical deflector so as to disperse multi-chromatic light in a direction opposite to that dispersed by said second acousto-optical deflector.
The present invention is further directed to a multi-photon excited fluorescence laser scanning microscope for forming a magnified image of a sample, said sample containing fluorescent molecules which radiate photons of a first characteristic energy, said multi-photon laser scanning microscope comprising (a) means for producing a scanning beam of ultrashort laser light pulses, said scanning beam producing means comprising (i) a laser source for providing a beam of ultrashort laser light pulses comprising photons of a second characteristic energy, wherein said second characteristic energy is less than said first characteristic energy and wherein the simultaneous absorption of a plurality of said photons of said second characteristic energy by said fluorescent molecules causes the fluorescence of said fluorescent molecules, (ii) a first acousto-optical deflector optically coupled to said laser source for scanning said beam along a first axis, (iii) a first spectrally dispersive element optically coupled to said first acousto-optical deflector, said first spectrally dispersive element being oriented relative to said first acousto-optical deflector so as to disperse multi-chromatic light in a direction opposite to that dispersed by said first acousto-optical deflector; (b) means for focusing said scanning beam to a focal point within said sample to produce an illumination intensity sufficiently high only at said focal point to produce molecular excitation and fluorescence of said sample by simultaneous absorption of at least two incident photons; (c) means for detecting the fluorescence produced by said sample; and (d) means for using the detected fluorescence to form a magnified image of the sample.
Preferably, the first spectrally dispersive element of the aforementioned microscope is constructed to disperse multi-chromatic light, for at least a portion of said multi-chromatic light, in an amount equal to that dispersed by said first acousto-optical deflector. The scanning beam producing means of the microscope described above preferably further comprises means for scanning the sample in a direction perpendicular to said first axis, said means for scanning the sample in a direction perpendicular to said first axis comprising a scanning mirror or a second acousto-optical deflector and a second spectrally dispersive element, said second spectrally dispersive element being oriented relative to said second acousto-optical deflector so as to disperse multi-chromatic light in a direction opposite to that dispersed by said second acousto-optical deflector.
The present invention is still further directed to a laser scanning microscope for forming a magnified image of a sample, the sample containing fluorophores, said laser scanning microscope comprising (a) means for producing a scanning beam of light pulses, said scanning beam producing means comprising (i) means for providing a beam of light pulses, said light pulses being of a wavelength suitable to excite said fluorophores, (ii) a first acousto-optical deflector optically coupled to said beam providing means for scanning said beam along a first axis, (iii) a first spectrally dispersive element optically coupled to said first acousto-optical deflector, said first spectrally dispersive element being oriented relative to said first acousto-optical deflector so as to disperse multi-chromatic light in a direction opposite to that dispersed by said first acousto-optical deflector; (b) means for focusing said scanning beam at a focal point within said sample; (c) means for detecting the fluorescence produced by said sample; and (d) means for using the detected fluorescence to form a magnified image of the sample.
The present invention is yet further directed to a method of imaging a sample using multi-harmonic generation laser scanning microscopy, said method comprising the steps of (a) providing a sample, the sample containing molecules having the appropriate nonlinear susceptibility; (b) producing a scanning beam of ultrashort laser light pulses, said scanning beam being produced by (i) providing a beam of ultrashort light pulses comprising photons of a first wavelength capable of interacting with said molecules having the appropriate nonlinear susceptibility to create, by multi-harmonic generation, photons of a second wavelength, (ii) passing said beam through a spectrally dispersive element, and (iii) deflecting said beam using an acousto-optical deflector, (iv) wherein said spectrally dispersive element is oriented to disperse multi-chromatic light in a direction opposite to that dispersed by said acousto-optical deflector; (c) focusing said scanning beam at a focal point within said sample to produce an illumination intensity sufficiently high only at said focal point to generate, by multi-harmonic generation, photons of said second wavelength; (d) detecting the photons of said second wavelength emitted from said sample; and (e) using the detected photons of said second wavelength to form an image of the sample.
Preferably, the spectrally dispersive element used in the aforementioned method is constructed to disperse multi-chromatic light, for at least a portion of said multi-chromatic light, in an amount equal to that dispersed by said acousto-optical deflector. The scanning beam producing step described above preferably further comprises scanning the sample in a direction perpendicular to said first axis, said scanning in a direction perpendicular to said first axis comprising the use of a scanning mirror or a second acousto-optical deflector and a second spectrally dispersive element, said second spectrally dispersive element being oriented relative to said second acousto-optical deflector so as to disperse multi-chromatic light in a direction opposite to that dispersed by said second acousto-optical deflector.
The present invention is further directed to a multi-harmonic generation laser scanning microscope for forming a magnified image of a sample, the sample containing molecules having the appropriate nonlinear susceptibility, said multi-harmonic generation laser scanning microscope comprising (a) means for producing a scanning beam of ultrashort laser light pulses, said scanning beam producing means comprising (i) a laser source for providing a beam of ultrashort light pulses comprising photons of a first wavelength capable of interacting with said molecules having the appropriate nonlinear susceptibility to create, by multi-harmonic generation, photons of a second wavelength, (ii) a first acousto-optical deflector optically coupled to said laser source for scanning said beam along a first axis, (iii) a first spectrally dispersive element optically coupled to said first acousto-optical deflector, said first spectrally dispersive element being oriented relative to said first acousto-optical deflector so as to disperse multi-chromatic light in a direction opposite to that dispersed by said first acousto-optical deflector; (b) means for focusing said scanning beam at a focal point within said sample to produce an illumination intensity sufficiently high only at said focal point to generate, by multi-harmonic generation, photons of said second wavelength; (c) means for detecting the photons of said second wavelength emitted from said sample; and (d) means for using the detected photons of said second wavelength to form an image of the sample.
Preferably, the first spectrally dispersive element of the aforementioned microscope is constructed to disperse multi-chromatic light, for at least a portion of said multi-chromatic light, in an amount equal to that dispersed by said first acousto-optical deflector. The scanning beam producing means of the microscope described above preferably further comprises means for scanning the sample in a direction perpendicular to said first axis, said means for scanning the sample in a direction perpendicular to said first axis comprising a scanning mirror or a second acousto-optical deflector and a second spectrally dispersive element, said second spectrally dispersive element being oriented relative to said second acousto-optical deflector so as to disperse multi-chromatic light in a direction opposite to that dispersed by said second acousto-optical deflector.
Additional objects, features, aspects and advantages of the present invention will be set forth, in part, in the description which follows and, in part, will be obvious from the description or may be learned by practice of the invention. In the description, reference is made to the accompanying drawings which form a part thereof and in which are shown by way of illustration specific embodiments for practicing the invention. These embodiments will be described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that structural changes may be made without departing from the scope of the invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is best defined by the appended claims.